Reduction of brain stem pathology and transient amelioration of early cognitive symptoms in transgenic mice treated with a monoclonal antibody against α-synuclein oligomers/protofibrils.

Immunotherapy against alpha-synuclein (α-syn) is a promising novel treatment strategy for Parkinson's disease (PD) and related α-synucleinopathies. We have previously shown that systemic treatment with the monoclonal oligomer/protofibril-selective antibody mAb47 targeting cytotoxic α-syn leads to reduced central nervous system levels of such species as well as an indication of reduced late-stage symptoms in aged (Thy-1)-h[A30P] α-syn transgenic mice. Here, we performed an early-onset long-term treatment study with this antibody to evaluate effects on brain pathology and behavioral outcomes in the same mouse model. Compared to the placebo group, the treatment strongly reduced phosphorylated α-syn (pS129 α-syn) pathology in the upper brain stem. Moreover, a preserved recognition memory and risk assessment behavior could be seen in antibody-treated mice at six months of age, even although these effects were no longer significant at eleven months of age. Importantly, no evidence of inflammatory responses or other potential toxic effects was seen with the treatment. Taken together, this study supports the strategy to target α-syn oligomers/protofibrils with monoclonal antibodies to counteract early symptoms and slow down the progression of PD and other α-synucleinopathies.

Trichloroethylene and its metabolite TaClo lead to degeneration of substantia nigra dopaminergic neurones: Effects in wild type and human A30P mutant α-synuclein mice.

Parkinson's disease (PD) is characterised pathologically by degeneration of the dopaminergic (DA) neurones of the substantia nigra pars compacta (SNpc) and the presence of α-synuclein containing Lewy body inclusions. Trichloroethylene (TCE) has been suggested as a potential environmental chemical that may contribute to the development of PD, via conversion to the neurotoxin, 1-Trichloromethyl-1,2,3,4-tetrahydro-ß-carboline (TaClo). We investigated the effect of an 8 week exposure to TCE or TaClo on wild type and, as an experimental model of PD, A30P mutant α-synuclein overexpressing mice using a combination of behaviour and pathology. TCE or TaClo exposure caused significant DA neuronal loss within the SNpc in both wild type and transgenic mice. Cell numbers were lower in A30P animals than wild type, however, no additive effect of TCE or TaClo exposure and A30P overexpression was found. TCE or TaClo did not appear to lead to acceleration of motor or cognitive deficits in either wild type or A30P mutant mice, potentially because of the modest reductions of DA neuronal number in the SNpc. Our results do however suggest that TCE exposure could be a possible factor in development of PD like changes following exposure.

Metformin lowers Ser-129 phosphorylated α-synuclein levels via mTOR-dependent protein phosphatase 2A activation.

Phospho-Ser129 α-synuclein is the modified form of α-synuclein that occurs most frequently within Parkinson's disease pathological inclusions. Here we demonstrate that the antidiabetic drug metformin significantly reduces levels of phospho-Ser129 α-synuclein and the ratio of phospho-Ser129 α-synuclein to total α-synuclein. This effect was documented in vitro in SH-SY5Y and HeLa cells as well as in primary cultures of hippocampal neurons. In vitro work also elucidated the mechanisms underlying metformin's action. Following metformin exposure, decreased phospho-Ser129 α-synuclein was not strictly dependent on induction of AMP-activated protein kinase, a primary target of the drug. On the other hand, metformin-induced phospho-Ser129 α-synuclein reduction was consistently associated with inhibition of mammalian target of rapamycin (mTOR) and activation of protein phosphatase 2A (PP2A). Evidence supporting a key role of mTOR/PP2A signaling included the finding that, similar to metformin, the canonical mTOR inhibitor rapamycin was capable of lowering the ratio of phospho-Ser129 α-synuclein to total α-synuclein. Furthermore, no decrease in phosphorylated α-synuclein occurred with either metformin or rapamycin when phosphatase activity was inhibited, supporting a direct relationship between mTOR inhibition, PP2A activation and protein dephosphorylation. A final set of experiments confirmed the effectiveness of metformin in vivo in wild-type C57BL/6 mice. Addition of the drug to food or drinking water lowered levels of phospho-Ser129 α-synuclein in the brain of treated animals. These data reveal a new mechanism leading to α-synuclein dephosphorylation that could be targeted for therapeutic intervention by drugs like metformin and rapamycin.

DJ-1-deficient mice show less TH-positive neurons in the ventral tegmental area and exhibit non-motoric behavioural impairments.

Loss of function of DJ-1 (PARK7) is associated with autosomal recessive early-onset Parkinson's disease (PD), one of the major age-related neurological diseases. In this study, we extended former studies on DJ-1 knockout mice by identifying subtle morphological and behavioural phenotypes. The DJ-1 gene trap-induced null mutants exhibit less dopamine-producing neurons in the ventral tegmental area (VTA). They also exhibit slight changes in behaviour, i.e. diminished rearing behaviour and impairments in object recognition. Furthermore, we detected subtle phenotypes, which suggest that these animals compensate for the loss of DJ-1. First, we found a significant upregulation of mitochondrial respiratory enzyme activities, a mechanism known to protect against oxidative stress. Second, a close to significant increase in c-Jun N-terminal kinase 1 phosphorylation in old DJ-1-deficient mice hints at a differential activation of neuronal cell survival pathways. Third, as no change in the density of tyrosine hydroxylase (TH)-positive terminals in the striatum was observed, the remaining dopamine-producing neurons likely compensate by increasing axonal sprouting. In summary, the present data suggest that DJ-1 is implicated in major non-motor symptoms of PD appearing in the early phases of the disease-such as subtle impairments in motivated behaviour and cognition-and that under basal conditions the loss of DJ-1 is compensated.

Nuclear and neuritic distribution of serine-129 phosphorylated alpha-synuclein in transgenic mice.

Parkinson's disease and dementia with Lewy bodies are very frequent neurological disorders of the elderly. Mutations in the alpha-synuclein (alphaSYN) gene cause Parkinson's disease, often associated with dementia. Neuropathologically these diseases are characterized by the presence of Lewy bodies and Lewy neurites, intraneuronal inclusions mostly composed of alphaSYN protein fibrils. Moreover, alphaSYN is phosphorylated at S129 (phospho-serine-129 [PSer129]) in neuropathological lesions. Using our (Thy1)-[A30P]alphaSYN transgenic mouse model that develops age-dependent impairment in fear conditioning behavior, we investigated PSer129 immunostaining in the brain. We found distinct staining patterns using new, sensitive monoclonal antibodies. Somal and nuclear PSer129 immunoreactivity increased with age in hippocampal and cortical areas as well as the lateral/basolateral amygdalar nuclei and was present also in young, pre-symptomatic mice, but not wild-type controls. The tendency of PSer129 immunostaining to accumulate in the nucleus was confirmed in cell culture. (Thy1)-[A30P]alphaSYN transgenic mice further developed age-dependent, specific neuritic/terminal alphaSYN pathology in the medial parts of the central amygdalar nucleus and one of its projection areas, the lateral hypothalamus. Interestingly, this type of PSer129 neuropathology was thioflavine S negative, unlike the Lewy-like neuropathology present in the brain stem of (Thy1)-[A30P]alphaSYN mice. Thus, alphaSYN becomes phosphorylated in distinct parts of the brain in this alpha-synucleinopathy mouse model, showing age-dependent increases of nuclear PSer129 in cortical brain areas and the formation of neuritic/terminal PSer129 neuropathology with variable amyloid quality within the fear conditioning circuitry and the brain stem.

Suppression of MAP kinases inhibits microglial activation and attenuates neuronal cell death induced by alpha-synuclein protofibrils.

Alpha-Synuclein (alpha-Syn) accounts, as a major component of Lewy bodies (LB), for the filamentous deposits in many cases of neurodegenerative diseases. Yet, little is known about the molecular mechanisms of neuronal loss in these diseases. The correlation between alpha-Syn oligomerization/aggregation and pathologies raises the key question of which molecular form of alpha-Syn (i.e. monomeric alpha-Syn, protofibrils or mature fibrils) represents the damage-inducing culprit in the scenario of synucleinopathies. We show that human alpha-Syn protofibrils (PFs) are potent activators of parallel proinflammatory signalling pathways (p38 and ERK1/2 MAP kinases and NF-kappaB) in microglial cells in vitro. Furthermore, stereotactic injection of alpha-Syn PFs into the substantia nigra of adult rats leads to a profound activation of microglia and adjacent neuronal cell loss, which can be attenuated by the MAP kinase inhibitor semapimod. We propose that the neurodegenerative process of alpha-synucleinopathies involves microglial activation through alpha-Syn released or extruded from cells with pathogenic alpha-Syn metabolism. Compounds that inhibit the MAPK/NF-kappaB pathways might be a promising pharmacological strategy for the treatment of the inflammatory component of synucleinopathies including PD.

Parkin interacts with the proteasome subunit alpha4.

Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.

Selective insolubility of alpha-synuclein in human Lewy body diseases is recapitulated in a transgenic mouse model.

alpha-Synuclein (alpha-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinson's disease (PD) and LB dementias. alpha-SYN forms LB-like fibrils in vitro, in contrast to its homologue beta-SYN. Here we have investigated the solubility of SYNs in human LB diseases and in transgenic mice expressing human wild-type and PD-associated mutant [A30P]alpha-SYN driven by the brain neuron-specific promoter, Thy1. Distinct alpha-SYN species were detected in the detergent-insoluble fractions from brains of patients with PD, dementia with LBs, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease). Using the same extraction method, detergent-insolubility of human alpha-SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse alpha-SYN nor beta-SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic beta-SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of alpha-SYN are absent in beta-SYN. In conclusion, the specific accumulation of detergent-insoluble alpha-SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.

Sensitivity to MPTP is not increased in Parkinson's disease-associated mutant alpha-synuclein transgenic mice.

Environmental and genetic factors that contribute to the pathogenesis of Parkinson's disease are discussed. Mutations in the alpha-synuclein (alphaSYN ) gene are associated with rare cases of autosomal-dominant Parkinson's disease. We have analysed the dopaminergic system in transgenic mouse lines that expressed mutant [A30P]alphaSYN under the control of a neurone-specific Thy-1 or a tyrosine hydroxylase (TH) promoter. The latter mice showed somal and neuritic accumulation of transgenic [A30P]alphaSYN in TH-positive neurones in the substantia nigra. However, there was no difference in the number of TH-positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum between these transgenic mice and non-transgenic littermates. To investigate whether forced expression of [A30P]alphaSYN increased the sensitivity to putative environmental factors we subjected transgenic mice to a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) regimen. The MPTP-induced decrease in the number of TH-positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum did not differ in any of the [A30P]alphaSYN transgenic mouse lines compared with wild-type controls. These results suggest that mutations and forced expression of alphaSYN are not likely to increase the susceptibility to environmental toxins in vivo.

The emerging utility of animal models of chronic neurodegenerative diseases.

The two most common neurodegenerative diseases are Alzheimer's disease (AD) and Parkinson's disease (PD). The symptoms are caused by the initially selective degeneration of neuronal subpopulations involved in memory (AD) or movement control (PD). The cause of both diseases is unknown, but ageing is an inevitable risk factor. The identification of disease-associated genes was a breakthrough for the understanding of molecular mechanisms of neurodegeneration and has provided the basis for the establishment of cell culture and animal model systems, instrumental for target validation and drug screening. Familial AD is caused by mutations in the beta-amyloid precursor protein (betaAPP) and in the gene products responsible for its proteolytic processing, namely the presenilins. Transgenic mice expressing these mutant genes develop characteristic AD plaques in an age-dependent manner. A reduction of plaque burden and amelioration of cognitive decline in these animals was recently achieved by vaccination with amyloid beta-protein fibrils. The other hallmark lesion of AD, the neurofibrillary tangle, has been modelled recently in transgenic mice expressing mutant tau protein linked to frontotemporal dementia. PD is characterised by intraneuronal cytoplasmic deposits (Lewy bodies) of the PD-associated gene product alpha-synuclein. Transgenic expression of alpha-synuclein recreated hallmark features of PD in mice and fruit flies, establishing alpha-synuclein as PD-causing drug target. Moreover, environmental risk factors such as the pesticide rotenone have been used successfully to generate rodent models of PD. Lesion models of PD are being exploited for the development of experimental gene therapy and transplantation approaches.

Lewy body variant of Alzheimer's disease: alpha-synuclein in dystrophic neurites of A beta plaques.

The contribution of alpha-synuclein accumulation in Alzheimer's disease (AD) plaques is currently a matter of scientific debate. In the present study antisera against the N- and C-terminus, the full-length protein and the central so-called non-amyloid component (NAC) domain of the alpha-synuclein protein were used to address this question in brains of cases with typical AD and of cases with the Lewy body (LB) variant of AD. In typical AD cases, none of the antisera revealed evidence for co-accumulation of alpha-synuclein with extracellular A beta peptides in plaques or in dystrophic neurites decorating the plaque core. Interestingly, cases with mixed pathology of the LB variant of AD revealed accumulation of alpha-synuclein in LBs and in dystrophic neurites of A beta plaques.

Subcellular localization of wild-type and Parkinson's disease-associated mutant alpha -synuclein in human and transgenic mouse brain.

Mutations in the alpha-synuclein (alphaSYN) gene are associated with rare cases of familial Parkinson's disease, and alphaSYN is a major component of Lewy bodies and Lewy neurites. Here we have investigated the localization of wild-type and mutant [A30P]alphaSYN as well as betaSYN at the cellular and subcellular level. Our direct comparative study demonstrates extensive synaptic colocalization of alphaSYN and betaSYN in human and mouse brain. In a sucrose gradient equilibrium centrifugation assay, a portion of betaSYN floated into lower density fractions, which also contained the synaptic vesicle marker synaptophysin. Likewise, wild-type and [A30P]alphaSYN were found in floating fractions. Subcellular fractionation of mouse brain revealed that both alphaSYN and betaSYN were present in synaptosomes. In contrast to synaptophysin, betaSYN and alphaSYN were recovered from the soluble fraction upon lysis of the synaptosomes. Synaptic colocalization of alphaSYN and betaSYN was directly visualized by confocal microscopy of double-stained human brain sections. The Parkinson's disease-associated human mutant [A30P]alphaSYN was found to colocalize with betaSYN and synaptophysin in synapses of transgenic mouse brain. However, in addition to their normal presynaptic localization, transgenic wild-type and [A30P]alphaSYN abnormally accumulated in neuronal cell bodies and neurites throughout the brain. Thus, mutant [A30P]alphaSYN does not fail to be transported to synapses, but its transgenic overexpression apparently leads to abnormal cellular accumulations.

Combined assessment of tau and neuronal thread protein in Alzheimer's disease CSF.

OBJECTIVE: Comparative study of CSF levels of tau and AD7C-neuronal thread protein (NTP) in patients with AD and control subjects. BACKGROUND: AD is characterized by neurofibrillary tangles composed of the abnormally hyperphosphorylated microtubule-associated protein tau. AD7C-NTP is a proposed AD marker expressed at early stages of neurofibrillary degeneration. METHODS: Enzyme-linked immunosorbent assays specific for tau and AD7C-NTP. CSF samples were obtained from 35 demented patients (25 with antemortem clinical diagnosis of probable AD, 5 with neuropathologic diagnosis of definite AD, 5 with Lewy body pathology), 29 nondemented patients with PD, and 16 elderly healthy control subjects. Receiver operating characteristics (ROC) and multivariate discriminant analysis for AD versus controls. Correlational analysis of CSF tau and AD7C-NTP and of each marker with Mini-Mental State Examination (MMSE) scores was performed. RESULTS: Levels of both tau and AD7C-NTP were significantly elevated in the AD patients compared with control subjects. ROC analysis showed that CSF tau distinguished between patients with AD and nondemented control subjects with 63% sensitivity and 89% specificity, AD7C-NTP with 70% sensitivity and 87% specificity. Combined evaluation of both markers with discriminant analysis raised the specificity to 93% at a 63% sensitivity level. Both markers positively correlated with each other within the AD group, but not among control subjects. CSF levels of AD7C-NTP, but not of tau, showed a small but significant inverse correlation (r = -0.43) with MMSE scores of AD patients. CONCLUSIONS: CSF levels of tau and AD7C-NTP may be useful biomarkers for AD.

Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein.

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.

Physiology and pathophysiology of alpha-synuclein. Cell culture and transgenic animal models based on a Parkinson's disease-associated protein.

The 15-20 kDa synuclein (SYN) phosphoproteins are abundantly expressed in nervous tissue. Members of the family include alpha- and beta-SYN, and the more distantly related gamma-SYN and synoretin. SYN genes have been identified in Torpedo, canary, and several mammalian species, indicating an evolutionary conserved role. Expression of alpha-SYN was found to be modulated in situations of neuronal remodeling, namely, songbird learning and after target ablation of dopaminergic striatonigral neurons in the rat. The presynaptic localization of alpha-SYN is further supportive of a direct physiological role in neuronal plasticity. The extensive synaptic co-localization of alpha- and beta-SYN might indicate functional redundancy of these highly homologous synucleins. However, alpha-SYN was the only family member identified in Lewy bodies and cytoplasmic inclusions characteristic for multiple system atrophy. Moreover, alpha-SYN was genetically linked to familial Parkinson's disease. The two Parkinson's disease-associated mutations accelerated the intrinsic aggregation property of alpha-SYN in vitro. Post-translational modifications, such as phosphorylation and proteolysis, and/or interaction with other proteins, might regulate alpha-SYN fibril formation in vivo. Cytoskeletal elements and signal transduction intermediates have been recently identified as binding partners for alpha-SYN. Preliminary data available from transgenic mice suggest that (over)expressed human alpha-SYN proteins are less efficiently cleared from the neuronal cytosol. Thus, Parkinson's disease-associated mutations might perturb axonal transport, leading to somal accumulation of alpha-SYN and eventually Lewy body formation.

Neurons and mechanisms of neuronal death in neurodegenerative diseases: a brief review.

BACKGROUND AND PURPOSE: Degenerative diseases of the central nervous system are a heterogenous group of slowly progressive disorders. A common feature of this group, which includes Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, is gradual loss of specific populations of neurons. METHODS: A series of reports about neurodegenerative diseases and their relevant animal models, as well as a brief overview of the normal neuron and mechanisms of neuronal degeneration and death, is presented. CONCLUSION: Study of the aforementioned animal models, spontaneously occurring and experimentally induced, have provided important insights into the pathogenesis of these disorders and the development of effective therapeutic strategies.

Phosphatidylcholine-specific phospholipase inhibitor D609 differentially affects MAP kinases and immediate-early genes in PC12 cells.

The effects of tricyclodecan-9-yl xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipases, on PC12 cells were investigated. D609 repressed nerve growth factor (NGF)-mediated induction of c-fos mRNA with an IC50 approximately 50 microg/ml. Interestingly, maximal c-fos-suppressing doses of D609 did not affect activity of extracellular signal-regulated kinases. Surprisingly, D609 enhanced the extracellular acidification rate of PC12 cells, even in the absence of NGF. D609 alone induced c-jun mRNA with the same potency as it repressed the NGF-induced expression of c-fos. Like NGF, D609 alone induced c-jun even in the presence of dominant-negative Ras. Immediate-early induction of c-jun mRNA by NGF and D609 was abrogated by pretreatment with the kinase inhibitor olomoucine. Jun kinase, which is inhibited by olomoucine, was found to be activated by D609. Thus, D609 might induce c-jun in PC12 cells as a consequence of Jun kinase activation through a Ras-independent pathway. Under the same conditions, D609 repressed NGF-mediated induction of c-fos mRNA.